Different CprABC amino acid sequences affect nisin A susceptibility in Clostridioides difficile isolates

Clinical isolates of Clostridioides difficile sometimes exhibit multidrug resistance and cause diarrhea after antibiotic administration. Metronidazole and vancomycin are often used as therapeutic agents, but resistance to these antibiotics has been found clinically. Therefore, the development of alternative antimicrobial agents is needed. Nisin A, produced by Lactococcus lactis, has been demonstrated to be effective against C. difficile infection. In this study, we evaluated the susceptibility of 11 C. difficile clinical isolates to nisin A and found that they could be divided into 2 groups: high and low susceptibility. Since CprABC and DltDABC, which are responsible for nisin A efflux and cell surface charge, respectively, have been reported to be related to nisin A susceptibility, we investigated the expression of cprA and dltA among the 11 strains. cprA expression in all strains was induced by nisin A, but dltA expression was not. The expression levels of both genes did not correlate with nisin A susceptibility in these clinical isolates. To evaluate cell surface charge, we performed a cytochrome C binding assay and found no relationship between charge and nisin A susceptibility. Then, we determined the whole genome sequence of each clinical isolate and carried out phylogenetic analysis. The 11 isolates separated into two major clusters, which were consistent with the differences in nisin A susceptibility. Furthermore, we found common differences in several amino acids in the sequences of CprA, CprB, and CprC between the two clusters. Therefore, we speculated that the different amino acid sequences of CprABC might be related to nisin A susceptibility. In addition, C. difficile strains could be divided in the same two groups based on susceptibility to epidermin and mutacin III, which are structurally similar to nisin A. These results suggest that genotypic variations in C. difficile strains confer different susceptibilities to bacteriocins.


Ethics
All the clinical C. difficile isolates were anonymized prior to obtaining by us, and there was no information about patients. The Ethical Committee for Epidemiology of Hiroshima University reviewed our application and approved it (E2019-1941).

Susceptibility test
Two methods were used for the evaluation of antibacterial activity. To measure the MIC value of nisin A (Sigma-Aldrich, St. Louis, USA), the microdilution method was used as described elsewhere [23]. Bacitracin (Fujifilm Wako chemicals, Osaka, Japan), vancomycin (Sigma-Aldrich), Metronidazole (Fujifilm Wako chemicals), ampicillin (Nacalai Tesque, Kyoto, Japan), chloramphenicol (Wako chemicals), gentamicin (Nacalai Tesque), ofloxacin (Sigma-Aldrich) and imipenem (Fujifilm Wako chemicals) were also used for MIC evaluations. To assess the antibacterial activity of the bacteriocins, a direct assay was performed by a method described elsewhere [23]. An overnight culture (3 μl) of the bacteriocin-producing strain, as indicator bacteria, was spotted on a TSA plate and cultured at 37˚C for 24 h. Then, 3.5 ml of prewarmed BHI soft agar (0.75%) containing C. difficile cells (10 8 cells/ml) was poured over the TSA plate. The plates were incubated anaerobically at 37˚C for 24 h. Then, the diameters of the growth inhibitory zones were measured in three directions. Three independent experiments were performed, and the average diameters were calculated.

Cytochrome c binding assay
A cytochrome c binding assay was performed to evaluate the cell surface charge as described elsewhere [33]. Bacterial cells in the exponential phase were collected and suspended in 10 mM sodium phosphate buffer (pH 6.8) by adjusting to a concentration of 10 9 cells/ml. Cytochrome c (Sigma Aldrich) was added to the bacterial suspensions at a final concentration of 250 μg/ml. After 10 min of incubation at room temperature, each bacterial suspension was centrifuged at 15 000 rpm for 5 min. The absorbance of the supernatant at 530 nm was measured. By comparing the absorbance values with and without bacterial cells, the absorption ratio was calculated to reflect the bacterial surface charge.

Gene expression analysis by quantitative PCR
Quantitative PCR (qPCR) was performed to investigate the expression of genes involved in bacteriocin susceptibility. Overnight cultures (500 μl each) of the C. difficile strains were inoculated into 5 ml of BHI broth and then grown at 37˚C under anaerobic conditions. After 5 h (0.2 by optical density at 660 nm), nisin A (final concentration: 16 μg/ml) was added to each bacterial culture. After 30 min, the bacterial cells were collected. RNA extraction, cDNA synthesis and qPCR were performed as described previously [23]. The primers used in this study are listed in S1 Table. The expression of each gene was quantified against 16S rRNA expression.

Effect of nisin A on germination
Prior to the germination assay, spores were purified by a method described elsewhere [34]. To evaluate the effect of nisin A on germination, we selected 2 strains with different DNA type, JCM5243 (Group 1) and H39 (Group 2). C. difficile JCM5243 and H39 were grown in BHI broth with 0.5% yeast extract and 0.1% L-cysteine at 37˚C under anaerobic conditions. The overnight culture was then plated on BHI agar with 0.1% L-cysteine and anaerobically incubated at 37˚C. After 7 days of incubation, the plate was placed at 4˚C for 24 h. Bacterial cells were scraped from the agar plate and washed with phosphate-buffered saline (PBS) 2 times. Then, the cells were suspended in PBS containing 125 mM Tris, 200 mM EDTA and 0.3 mg/ml proteinase K and incubated aerobically with gentle agitation at 37˚C for 2 h. Then, the spores were washed with sterilized water 10 times. The spore solution was stored for up to 1 year at 4˚C prior to use. We confirmed the number of spores in each experiment before use.
Before the germination assay, spore stocks were incubated at 65˚C for 30 min. Spores (10 5 spores) were added to BHI broth with or without 0.1% taurocholate (an inducer for germination) in the presence or absence of nisin A (32 or 512 μg/ml). The spore solution was incubated anaerobically at 37˚C for 30 min. Then, appropriate dilutions were plated on BHI agar with 0.1% taurocholate and incubated anaerobically at 37˚C for 2 days. Colony-forming units (CFU) were counted. Three independent experiments were performed.

Genome sequence analysis of the C. difficile isolates
To perform whole-genome sequencing of the C. difficile isolates, DNA was extracted from each isolate. Genomic DNA was purified from the lysate using AMPure XP beads (Beckman Coulter, USA). DNA libraries were prepared as described previously [35]. Whole-genome sequencing (WGS) of C. difficile isolates was performed using the Illumina MiSeq sequencing platform, followed by annotation with Rapid Annotation using Subsystem Technology (RAST) version 2.0 [36]. A phylogenetic tree was constructed using the CSI Phylogeny 1.4 pipeline available from the Center for Genomic Epidemiology (Lungby, Denmark) for SNP calling and then annotated using the iTOL web-based tool [37]. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.
We also compared the 11 clinical genomes with 50 C. difficile genomes that are available in the NCBI database. Fifty genomes were randomly selected from the database. Then, we performed phylogenetic tree analysis and compared the amino acid sequences of CprA, B and C among the 61 genomes, including 11 genomes sequenced in this study.

Accession number
The genome data of the C. difficile isolates used in this study have been deposited into the DDBJ Sequence Read Archive (DRA) accession number DRA014785 under the BioProject accession no. PRJDB14281.

Susceptibility of C. difficile to nisin A
The susceptibility to nisin A varied among the 11 clinical C. difficile isolates ( Table 2). The MIC values ranged from 2 to 64 μg/ml. Since the final nisin A concentration used in this study was 2.5%, the calculated MIC range was 0.05 to 1.6 μg/ml. The MIC value for each isolate correlated with the results of the direct assays, as the isolates with higher MIC values showed lower inhibitory zones ( Table 2,  Based on phylogenetic tree analysis, the 11 isolates were divided into 2 clusters designated Groups 1 and 2 (Fig 1). Group 1 included 6 isolates, showing nisin A MICs of 8 to 64 μg/ml,

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Nisin A susceptibility in C. difficile while Group 2 included 5 isolates, showing nisin A MICs of 2 to 8 μg/ml (Fig 2A). In the direct assay, the diameter length of Group 1 isolates showed smaller than that of Group 2 isolates (Fig 2B). In both assays, the susceptibility of the strains to nisin A in Group 1 was lower than that of Group 2.

Susceptibility of C. difficile strains to several bacteriocins and antibiotics
Since C. difficile displayed susceptibility to nisin A, we evaluated its susceptibility to several lantibiotics and non-lantibiotics (Table 3, Fig 3). The two groups divided by nisin A susceptibility also showed a significant difference in epidermin susceptibility (p = 0.0081, Fig 3). Susceptibility against mutacin I (p = 0.027), mutacin III (p = 0.024) and mutacin IIIb (p = 0.025) also tended to be higher in Group 2 than in Group 1. In contrast, no significant difference in

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susceptibility to mutacin IV (p = 0.35) was found between the two groups. Mundticin KS and lacticin Q showed no antibacterial activity of 9 strains and 7 strains among 12 strains, and we found no significant differences between the two groups (mundticin KS; p = 0.31, lacticin Q; p = 0.21). In addition, nukacin ISK-1 showed no antibacterial activity against all C. difficile   Fig 4). However, susceptibility of Groups 1 and 2 to the other antibacterial agents showed no significant differences.

Expression of cprA and dltD in C. difficile strains
Since cprA-C and dltA-D were previously reported to be associated with nisin A resistance [26][27][28], we investigated the expression of cprA and dltD in the strains with or without nisin A addition (Fig 5). All isolates showed that cprA expression was significantly induced by the addition of sub-MICs of nisin A, although the expression levels varied among the strains. The expression level in each strain was not separated by the groups classified defined by nisin A

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susceptibility. In the absence of nisin A, the cprA expression level was quite low in all strains. dltD expression was not induced by the addition of nisin A, and was very low in both the presence and absence of nisin A. The expression level of dltD among the 11 isolates showed no correlation with grouping.

Cytochrome c binding assay
We further investigated the cell surface charge because cell surface charge was associated with the susceptibility to cationic peptides including nisin A [28]. To evaluate the cell surface charge, a cytochrome binding assay was performed (Fig 6). Binding affinity was similar among all strains. There was no relationship between the binding affinity and nisin A susceptibility.

Comparison of CprA-C amino acid sequences
Since we found no correlation between nisin A susceptibility and the expression of cprA and dltD, we compared the amino acid sequences of CprA-C, which are intrinsic factors for nisin A resistance, among the strains (Fig 7). We found some consensus differences in several amino acids of CprA-C between Groups 1 and 2. The amino acid residues of CprA at positions 30 (serine), 70 (threonine), 116 (threonine) and 193 (glutamic acid) in Group 1 were different CprB and CprC in Group 1 also showed differences in 3 and 4 amino acids compared to those in Group 2, respectively. We further expanded our analysis by using 50 C. difficile genomes randomly selected from the NCBI database (Fig 8, S2 and S3 Figs). Although the clusters groups by phylogenetic tree analysis were further divided into several subgroups, the common differences in CprA-C amino acid patterns between Group 1 and Group 2 were also observed in the other genomes that originated somewhere else.

Effect of nisin A on C. difficile spores and germinated cells
To know the susceptibility to nisin A on spores and germinated cells in Group 1 and 2, we investigated the susceptibility in JCM5243 (Group 1) and H39 (Group 2). In the presence of 0.1% taurocholate, the addition of nisin A (32 or 512 μg/ml) for 30 min drastically reduced the number of cells of both C. difficile strains JCM5243 and H39 (Fig 9). In particular, nisin A at 512 μg/ml completely killed the bacterial cells of both strains. In the absence of 0.1% taurocholate, the number of spores in JCM5243 (MIC: 32 μg/ml) in the presence of 32 and 512 μg/ml showed 10.8.0% and 12.5% reduction, respectively, while the number of spores in H39 (MIC: 4 μg/ml) in the presence of 32 and 512 μg/ml showed 24.0% and 87.2% reduction, respectively.

Discussion
In this study, we found that nisin A susceptibility was different among C. difficile strains, which could be divided into 2 groups: high and low nisin A susceptibility. We also found that these groups correlated with the clusters based on whole-genome sequence phylogenetic analysis. Furthermore, it is noteworthy that the amino acid sequences of CprA-C were also divided into 2 groups corresponding to nisin A susceptibility and phylogenetic tree analysis. The cprA-C genes encode an ABC transporter, which consists of 2 permeases and 1 ATPase [26,27]. CprABC was demonstrated to be responsible for resistance to several lantibiotics, including nisin A. cprK, encoding histidine kinase, was located downstream of cprA-C, while cprR, encoding a response regulator, was separate from the cprA-K regions. CprKR regulates the expression of cprA-C in response to nisin A. We also found that the amino acid sequences of

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CprK and CprR were divided into 2 groups according to nisin A susceptibility (S4 Fig). However, cprA expression in all tested strains was increased by nisin A addition (Fig 5). The expression levels varied among the strains, but were not correlated with nisin A susceptibility. Therefore, we concluded that the different amino acid sequences of CprRK between the 2 groups were not related to nisin A susceptibility.
By comparing the amino acid sequence of CprA-C, we found that several amino acids (4, 3, and 4 amino acid differences in CprA, B and C, respectively) were commonly different in the 2

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groups. We further investigated these differences in CprA-C by randomly selecting 50 C. difficile genome datasets from the NCBI database. The number of clusters was increased after phylogenetic tree analysis, but these common amino acid differences were still found in each cluster (Fig 8, S2 and S3 Figs). Previously, we found amino acid sequence variations in Mut-FEG, an ABC transporter responsible for nisin A and mutacin I/III/IIIb resistance in Streptococcus mutans [38]. MutFEG was classified into 3 groups (MutFEGs α, β, and γ) based on amino acid sequence. Several amino acid substitutions of MutFEG altered susceptibility to nisin A, mutacin I and mutacin III/IIIb. Based on these results, we suggest that several amino acid differences in CprA-C are associated with susceptibility to nisin A, although we did not directly demonstrate whether these differences affect nisin A susceptibility by replacing cprA-C in each type of strain.
Since we found that the C. difficile strains separated into 2 groups based on nisin A susceptibility, we further investigated their susceptibility to other bacteriocins, including bacteriocins

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with structures similar to nisin A. The first and second ring structures of epidermin and mutacin I/III/IIIb showed a structure similar to that of nisin A [39] (S5 Fig). In particular, the secondary ring structures of epidermin and mutacin III/IIIb were identical to those of nisin A. In Table 3 and Fig 3, we found tendencies of susceptibility to epidermin and mutacin I/III/IIIb similar to that of nisin A, while we did not find the tendencies of susceptibility to class II bacteriocins. Suárez JM demonstrated that the expression of cprA was induced by several lantibiotics closely related to nisin A, including gallidermin and mutacin III [26]. From these results, it is speculated that the amino acid differences in CprA-C strongly contributed to susceptibility to nisin A and bacteriocins closely related to nisin A, although their contributions to the level of susceptibility were different.
In addition to CprA-C, the Dlt system in gram-positive bacteria including C. difficile has been reported to be involved in resistance to cationic antimicrobial peptides [28,[40][41][42]. Dlt is involved in alanine incorporation into teichoic or lipoteichoic acid. This incorporation adds a positive charge to these molecules, causing a shift to a weak negative charge on the bacterial cell surface. In this study, we investigated the expression of dltD in all strains in the presence and absence of nisin A, and we found that dltD expression had no correlation with nisin A susceptibility. In addition, the surface charge evaluated by the cytochrome C binding assay showed no correlation between cell surface charge and nisin A susceptibility. Therefore, we think that DltABCD is not associated with the different susceptibilities to nisin A found in this study.
In this study, we also found no correlation between antibiotic susceptibility and the 2 groups. However, we found that the vanXYG genes, which were reported to be involved in vancomycin susceptibility [43], were also only found in type 1 strains, but we did not find a difference in vancomycin susceptibility between the 2 groups. Previous reports also demonstrated that the presence of vanG did not contribute to vancomycin resistance [44]. It is interesting to note that the presence of antibiotic resistance genes is linked to C. difficile types defined by nisin A susceptibility, although the biological interaction between them remains unknown. Bacteriocin resistance is considered to be important for the survival of bacterial microbiota in certain environments, including the human body. Therefore, type 1 strains might have a strong ability to cause host infection compared to type 2 strains. Furthermore, we investigated the genes for CDT, toxin A (TcdA) and toxin B (TcdB) which are major virulence factors in C. difficile [1,2]. Interestingly, cdtAB genes were only found in Group I except H176 strain, while tcdAB genes were found in 4 (JCM1296 T , JCM5243, H153 and H197) of 6 strains in Group1 and 2 (H156 and H299) of 5 strains in Group 2. Therefore, the classification of susceptibility by nisin A is also shown to be related to the presence of the gene for CDT.
To know the different susceptibility to nisin A between Group 1 and Group 2 is found in spores or germinated cells, we also investigated the effect of nisin A on spores and germinated cells (Fig 9). Above the MIC of nisin A, germinated cells induced by 0.1% taurocholate were completely killed after 30 min. In contrast, in the absence of 0.1% taurocholate, strain JCM5243, which has low susceptibility to nisin A, did not show a reduction in viable spores after treatment with nisin A (32 or 512 μg/ml) for 30 min, but the H39 strain showed a reduction in viable spores after the addition of nisin A, although the spores were not completely killed. The surface of C. difficile spores consists of several layers, including the exosporium, coat, and cortex, which exhibit resistance to many environmental stimuli, such as heat, antibiotics and disinfectants [34]. Therefore, it seems that bacteriocins are not effective against C. difficile spores. However, treatment of spores with a high concentration of nisin A in the absence of 0.1% taurocholate reduced the cell (spore) numbers. This result could be explained by the fact that some portions of nisin A attach to the spore surface and show antibacterial activity against the highly susceptible H39 strain after spore germination induced by 0.1% taurocholate.
In conclusion, we first demonstrated the variation in nisin A susceptibility and some lantibiotics that are structurally similar to nisin A. This variation was considered to be due to the differences in CprA-C amino acid sequences that are responsible for nisin A resistance. Moreover, the different amino acid sequences of CprA-C are a result of genomic variations in C. difficile. Our results provide a new characteristic feature of C. difficile strains and give some considerations for the clinical application of nisin A against C. difficile infection.